Insoluble native collagen fibers and their use in cell aggregates and tissue constructs

ABSTRACT

A method of forming an insoluble native collagen fiber additive, includes soaking the decellularized tissue powder, which includes collagen fibers, in acidic or basic, salt solution, enzymatically dissociating the insoluble collagen fibers, and isolating insoluble native collagen fibers from the solution. The isolated insoluble collagen fibers can be used in cell aggregates and/or tissue constructs to provide three dimensional cell growth and/or adhesion.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No. 62/650,611, filed Mar. 30, 2018, the subject matter of which is incorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant Nos. R01AR063194 and T32HL134622, awarded by The National Institutes of Health. The United States government has certain rights to the invention.

TECHNICAL FIELD

The present invention generally relates to tissue engineering, bioactive factor delivery, and disease therapeutics, and more particularly relates to collagen fiber additives for use in cell aggregates and tissue constructs and methods of forming the cell aggregates and tissue constructs.

BACKGROUND

Emerged as a pioneering technology, three dimensional (3D) cell culture based tissue regeneration better recapitulates natural cell growing conditions compared to two dimensional (2D) culture. Various 3D culture systems have been developed and employed in tissue engineering, disease model studies and drug discoveries. These strategies are categorized into two major classes, including scaffold based and scaffold-free systems. Scaffold based systems, such as pre-fabricated scaffolds and assembled hydrogels, promote cell-matrix interactions. In such systems, cells are surrounded by dense natural or artificial extracellular matrices (ECMs) in all dimensions, resulting in enhanced response to physical/mechanical and chemical/biochemical cues from the environments. On the other hand, scaffold-free systems, for instance, spheroids and organoids, elevate cell-cell interactions, as cells are densely packed in aggregate forms. Therefore, cells are strongly affected by paracrine/juxtacrine from surrounding partners.

Despite substantial progress for 3D cell culture, significant challenges still remain. Natural growing environment usually possesses both strong cell-cell and cell-matrix interactions. However, in most 3D culture systems, the cost of elevating one type of interaction is to sacrifice the other. For example, in hydrogel systems, regardless of the density of embedded cells, individual cells are predominantly surrounded by hydrogel materials rather than other cells. Degradable hydrogels have to be utilized for the enhancement of cell-cell interactions. Populating cells into pre-fabricated and natural decellularized scaffolds rely on cell infiltration. Cell density inside these scaffolds greatly depends on scaffold pore sizes and depth. On the other hand, it is quite difficult to evenly introduce ECMs into densely packed cell aggregates. Developed examples include embedding cell aggregates in matrigel and similar materials. Unfortunately, neither of them provide satisfactory microenvironment for individual cells promoting both cell-cell and cell-matrix interactions.

SUMMARY

Embodiments described herein relate to insoluble native collagen fibers, methods of forming insoluble native collagen fibers, and their use in forming three dimensional (3D) cell aggregates and tissue constructs.

In some embodiments, a method of forming isolated insoluble native collagen fibers can include providing decellularized tissue powder that includes native collagen fibers. The tissue powder can be soaked in acidic or basic, salt solution for an amount of time effective to loosen the fibrous structure and remove nucleic acids and forming a slurry that includes insoluble collagen fibers. The insoluble collagen fibers in the slurry are then enzymatically dissociated to a degree that bundles of insoluble collagen fibers are loosened while native architecture of the insoluble collagen fibers is maintained with minimal generation of collagen fibril. The insoluble native collagen fibers can then be isolated from the dissociated slurry.

In some embodiments, the decellularized tissue powder can include decellularized ground skin powder. The acidic, salt solution can include, for example, 1 M sulfuric acid and 1 M NaCl. The insoluble collagen fibers can be enzymatically dissociated with pepsin.

In other embodiments, the insoluble native collagen fibers can be isolated by separating the insoluble native collagen fibers from dispersed collagen fibrils, soluble collagen, and non-collagen material in the dissociated slurry. The isolated insoluble native collagen fibers have an average diameter of about 1 μm to about 200 μm and average length of about 100 μm to about 3 mm.

In other embodiments, the insoluble native collagen fiber can be used in forming a cell aggregate or tissue construct. The cell aggregate can be scaffold-free and, optionally, self-assembled. The cell aggregate can include a population or plurality of cells and a plurality of dispersed insoluble native collagen fibers. The collagen fibers can support adhesion and growth of cells, provide mechanical strength to the cell aggregate as well as enhance cell aggregate size and/or thickness. The cell aggregate can be readily manipulated and formed into tissue constructs with defined architectures, such as sheets, oblate spheroids, spheroids, or potentially any other shape. The insoluble native collagen fibers can potentially enhance cell function, such as differentiation, and/or enhance or accelerate tissue formation.

In some embodiments, the insoluble native collagen fibers can include collagen type I, collagen type II, collagen type III, or combinations thereof. The insoluble native collagen fibers can be cross-linked and include cross-linked and/or non-cross-linked collagen fibrils.

In some embodiments, the cell aggregate can further include at least one bioactive agent. The at least one bioactive agent can be incorporated in or physically associated with the insoluble native collagen fibers.

In other embodiments, the bioactive agent can be incorporated in or physically associated with a plurality of nanoparticles and/or microparticles that are provided in cell aggregate. The bioactive agent can be spatially and/or temporally released with a defined release profile from the nanoparticles and/or microparticles.

In some embodiments, the nanoparticles and/or microparticles can be formed from a biocompatible and biodegradable polymer. In one example, the biodegradable polymer can include a hydrogel that comprises natural macromers, which can be cross-linked to vary the mechanical properties and/or degradation profile of the nanoparticles and/or microparticles.

In some embodiments, the bioactive agent can induce the formation of a cell sheet, graft, or structure. One example of the bioactive that can be used to induce the formation of a cell sheet, graft, or structure belongs to the Transforming Growth Factor family (TGF) (e.g., TGF-β1).

In other embodiments, the cell aggregate can include undifferentiated and/or substantially differentiated cells. The cell aggregate can include at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% cells based on the total volume of the cell aggregate.

In another embodiment, the cell can be an adult stem cell or cancer cell. The stem cells can be isolated from animal or human tissues. The cell used for the production of the cell aggregate can be autologous, allogeneic, or xenogeneic. In the embodiments, the cell is isolated from, but not limited to, tendon/ligament tissue, bone morrow, adipose tissue or dental pulp.

In still other embodiments, a heterogenous cell aggregate or tissue construct can be formed that includes defined regions or portion (e.g., layers) of differing or similar cell aggregate materials. The differing regions or portions of the heterogenous cell aggregate or tissue construct can be provided or formed with or without insoluble native collagen fibers and can have similar or different properties to vary the properties of the tissue construct for particular tissue engineering applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A-E) illustrate A) Scheme for collagen fiber preparation. B) Ground skin tissue powder before processing stained by Fast Green. C) extracted collagen fibers stained by Fast Green. D) AFM of an individual collagen fiber. E) Magnified image of (D) showing the structure of collagen fiber is entact.

FIGS. 2(A-F) illustrate A) Scheme of collagen fiber for cell growth. B) Fibroblast NIH/3T3 growing in collagen fibers after 1 day. C) Optical image showing NIH/3T3 attached to collagen fiber. D) HeLa cells growing in collagen fibers after 1 day. E) NIH/3T3 and HeLa proliferating in collagen fibers measured by DNA quantification. [[E]]F) hMSCs growing in collagen fibers up to 14 days. Bars: 100 μm.

FIG. 3 Co-culture of hMSCs and endothelial cells in collagen fibers, transwell, collagen gel and GelMA.

FIGS. 4(A-C) illustrate A) Shear modulus change after crosslinking and incubation at 37° C. B) Co-culture of hMSCs and endothelial cells in uncrosslinked and crosslinked collagen fibers for 2 weeks. C) Junction formation analysis for vasculogenesis.

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Commonly understood definitions of molecular biology terms can be found in, for example, Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Ed., Springer-Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present invention.

In the context of the present invention, the term “bioactive agent” can refer to any agent capable of promoting tissue formation, destruction, and/or targeting a specific disease state. Examples of bioactive agents can include, but are not limited to, chemotactic agents, various proteins (e.g., short term peptides, bone morphogenic proteins, collagen, glycoproteins, and lipoprotein), cell attachment mediators, biologically active ligands, integrin binding sequence, various growth and/or differentiation agents and fragments thereof (e.g., epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factors (VEGF), fibroblast growth factors (e.g., bFGF), platelet derived growth factors (PDGF), insulin-like growth factor (e.g., IGF-I, IGF-II) and transforming growth factors (e.g., TGF-β I-III), parathyroid hormone, parathyroid hormone related peptide, bone morphogenic proteins (e.g., BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-13, BMP-14), transcription factors, such as sonic hedgehog, growth differentiation factors (e.g., GDF5, GDF6, GDF8), recombinant human growth factors (e.g., MP52 and the MP-52 variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-1, CDMP-2, CDMP-3), small molecules that affect the upregulation of specific growth factors, tenascin-C, hyaluronic acid, chondroitin sulfate, fibronectin, decorin, thromboelastin, thrombin-derived peptides, heparin-binding domains, heparin, heparan sulfate, polynucleotides, DNA fragments, DNA plasmids, MMPs, TIMPs, interfering RNA molecules, such as siRNAs, oligonucleotides, proteoglycans, glycoproteins, glycosaminoglycans, and DNA encoding for shRNA.

As used herein, the terms “biodegradable” and “bioresorbable” may be used interchangeably and refer to the ability of a material (e.g., a natural polymer or macromer) to be fully resorbed in vivo. “Full” can mean that no significant extracellular fragments remain. The resorption process can involve elimination of the original implant material(s) through the action of body fluids, enzymes, cells, and the like.

As used herein, the term “function and/or characteristic of a cell” can refer to the modulation, growth, and/or proliferation of at least one cell, such as a progenitor cell and/or differentiated cell, the modulation of the state of differentiation of at least one cell, and/or the induction of a pathway in at least one cell, which directs the cell to grow, proliferate, and/or differentiate along a desired pathway, e.g., leading to a desired cell phenotype, cell migration, angiogenesis, apoptosis, etc.

As used herein, the term “polynucleotide” can refer to oligonucleotides, nucleotides, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, siRNA, miRNA, tRNA) of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acids, or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs). The term can also encompass nucleic acids (i.e., oligonucleotides) containing known analogues of natural nucleotides, as well as nucleic acid-like structures with synthetic backbones.

As used herein, the term “polypeptide” can refer to an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules. The term “polypeptide” can also include amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain any type of modified amino acids. The term “polypeptide” can also include peptides and polypeptide fragments, motifs and the like, glycosylated polypeptides, and all “mimetic” and “peptidomimetic” polypeptide forms.

As used herein, the term “cell” can refer to any progenitor cell, such as totipotent stem cells, pluripotent stem cells, and multipotent stem cells, as well as any of their lineage descendant cells, including more differentiated cells. The terms “stem cell” and “progenitor cell” are used interchangeably herein. The cells can derive from embryonic, fetal, or adult tissues. Examples of progenitor cells can include totipotent stem cells, multipotent stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells, neuronal stem cells, hematopoietic stem cells, pancreatic stem cells, cardiac stem cells, embryonic stem cells, embryonic germ cells, neural crest stem cells, kidney stem cells, hepatic stem cells, lung stem cells, hemangioblast cells, and endothelial progenitor cells. Additional exemplary progenitor cells can include de-differentiated chondrogenic cells, chondrogenic cells, cord blood stem cells, multi-potent adult progenitor cells, myogenic cells, osteogenic cells, tendogenic cells, ligamentogenic cells, adipogenic cells, and dermatogenic cells.

As used herein, the term “subject” can refer to any animal, including, but not limited to, humans and non-human animals (e.g., rodents, arthropods, insects, fish (e.g., zebrafish)), non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, ayes, etc.), which is to be the recipient of a particular treatment. Typically, the terms “patient” and “subject” are used interchangeably herein in reference to a human subject.

As used herein, the term “tissue” can refer to an aggregate of cells having substantially the same function and/or form in a multicellular organism. “Tissue” is typically an aggregate of cells of the same origin, but may be an aggregate of cells of different origins. The cells can have the substantially same or substantially different function, and may be of the same or different type. “Tissue” can include, but is not limited to, an organ, a part of an organ, bone, cartilage, skin, neuron, axon, blood vessel, cornea, muscle, fascia, brain, prostate, breast, endometrium, lung, pancreas, small intestine, blood, liver, testes, ovaries, cervix, colon, stomach, esophagus, spleen, lymph node, bone marrow, kidney, peripheral blood, embryonic, or ascite tissue.

As used herein, the terms “inhibit,” “silencing,” and “attenuating” can refer to a measurable reduction in expression of a target mRNA (or the corresponding polypeptide or protein) as compared with the expression of the target mRNA (or the corresponding polypeptide or protein) in the absence of an interfering RNA molecule of the present invention. The reduction in expression of the target mRNA (or the corresponding polypeptide or protein) is commonly referred to as “knock-down” and is reported relative to levels present following administration or expression of a non-targeting control RNA.

As used herein, the term “aggregate” can refer to a group or cluster comprising at least two or more cells (e.g., progenitor and/or differentiated cells).

As used herein, the term “population” can refer to a collection of cells, such as a collection of progenitor and/or differentiated cells.

As used herein, the term “differentiated” as it relates to the cells of the present invention can refer to cells that have developed to a point where they are programmed to develop into a specific type of cell and/or lineage of cells. Similarly, “non-differentiated” or “undifferentiated” as it relates to the cells of the present invention can refer to progenitor cells, i.e., cells having the capacity to develop into various types of cells within a specified lineage or in different lineages.

Embodiments described herein relate to isolated insoluble native collagen fibers, methods of forming insoluble native collagen fibers, and their use in forming cell aggregates and tissue constructs. The insoluble native collagen fibers can be made up of bundles of collagen fibrils, which in turn are ordered aggregates of collagen molecules. These native fibers are preferred for their architectural and mechanical characteristics, as they will provide enhanced physical properties when culturing with cells when compared to lower orders of collagen. The native collagen fibers are used as a structural element, wherein a source of collagen tissue, typically an allograft or xenograft source (e.g., porcine, bovine, caprine, piscine, ovine, etc.) has been prepared by chemically cleansing the tissue of non-collagenous substances while still maintaining the architectural structure of the collagen and then mechanically disrupting the resultant material, thus breaking it down into fibers which still maintain the unique architecture of the tissue from which it was sourced (i.e. hide, tendon, intestine, etc). Chemical reduction of the collagen to lower order forms (e.g., fibrillar, soluble) should be avoided or minimized, as it destroys the nativity of the collagen fibers. Importantly, the native collagen fibers are treated in a manner that preserves the native cross-links that are found in native collagen, and avoids disrupting the collagen structure, so as to avoid reducing the organizational level of the collagen to a fibrillar, soluble, or tropocollagen level. It is recognized that in the practice of the present invention, one may beneficially combine the fibers described above with other collagen forms, such as fibrillar or soluble collagen forms, however, it is important that there remain insoluble native collagen fibers, for the reasons described herein. The insoluble native collagen fibers as described herein are not to be confused with, reconstituted soluble or other lower forms of collagen, such as tropocollagen. These lower and reconstituted forms of collagen lose physical integrity and the natural binding sites during the unraveling of the collagen triple helix, which in turn leaves the base molecule with exposed telopeptides.

The isolated insoluble native collagen fibers can be derived from any collagen bearing tissue from an animal. The insoluble native collagen fibers can be allogenic or xenogenic. The insoluble native collagen fibers can be from skin, tendon, fascia, ligament, trachea, or organ collagen. In certain embodiments, the collagen is human collagen or other mammalian collagen (e.g., porcine, bovine, or ovine). The insoluble native collagen fibers can be sourced from any animal.

Presently, about twenty-eight distinct collagen types have been identified in vertebrates, including bovine, ovine, porcine, chicken, marine, and human sources. Generally, the collagen types are numbered by Roman numerals, and the chains found in each collagen type are identified by Arabic numerals. Detailed descriptions of structure and biological functions of the various different types of naturally occurring collagens are generally available in the art.

The insoluble native collagen fibers may have the same composition as in naturally occurring sources. Examples of sources of insoluble native collagen fibers include human or non-human (bovine, ovine, and/or porcine), as well as recombinant collagen or combinations thereof. Examples of suitable collagen includes, but are not limited to, collagen type I, collagen type II, collagen type III, collagen type IV, collagen type V, collagen type VI, collagen type VII, collagen type VIII, collagen type IX, collagen type X, collagen type XI, collagen type XII, collagen type XIII, collagen type XIV, collagen type XV, collagen type XVI, collagen type XVII, collagen type XVIII, collagen type XIX, collagen type XX, collagen type XXI, collagen type XXII, collagen type XVIII, collagen type XXIV, collagen type XXV, collagen type XXVI, collagen type XXVII, and collagen type XXVIII, or combinations thereof. Insoluble native collagen fibers may further or alternatively comprise hetero- and homo-trimers of any of the above-recited collagen types. In some embodiments, the insoluble native collagen fibers include hetero- or homo-trimers of human collagen type I, human collagen type II, human collagen type III, or combinations thereof.

In some embodiments, the insoluble native collagen fibers include all type I or substantially all is collagen type I, namely, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

The isolated insoluble native collagen fibers can be formed by providing decellularized tissue powder that includes native collagen fibers. The tissue can be from any collagen containing organ source, such as skin, fascia, intestine, tendon, bladder, trachea, heart, lung, liver, kidney, spleen and pancreas. In some embodiments, human compatible collagen, and xenograft collagen can be used if they can be rendered non-immunogenic. In some embodiments, the decellularized tissue powder can include decellularized ground skin powder. Sugars and other substances may be removed during processing such that at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, or at least 99% of them have been removed.

The tissue powder can be soaked in acidic or basic with salt solution for an amount of time effective to loosen the fibrous structure and remove nucleic acids and forming a slurry that includes insoluble collagen fibers. The acidic, salt solution can include, for example, 1 M sulfuric acid and 1 M NaCl, or 1 M sodium hydroxide and 1 M NaCl, or 1 M calcium hydroxide and 1 M NaCl.

The insoluble collagen fibers in the slurry are then enzymatically dissociated using, for example, pepsin to a degree that bundles of insoluble collagen fibers are loosened while native architecture of the insoluble collagen fibers is maintained with minimal generation of collagen fibril or tropocollagen.

The insoluble native collagen fibers can then be isolated from the dissociated slurry. by separating the insoluble native collagen fibers from dispersed collagen fibrils, soluble collagen, poorly dissociated bulky material, and non-collagen material in the dissociated slurry, using, for example, centrifugation techniques. The isolated insoluble native collagen fibers have an average diameter of about 1 μm to about 200 μm and average length of about 100 μm to about 3 mm, for example, an average diameter of about 5 μm to about 100 μm and average length of about 100 μm to about 1 mm.

In other embodiments, the insoluble native collagen fiber can be used in forming a cell aggregate or tissue construct. The cell aggregate can be scaffold-free and, optionally, self-assembled.

By scaffold-free, it is meant the cells are not seeded in a natural or artificial continuous polymer matrix scaffold that defines the area or volume or at least a portion of the area or volume of the cell aggregate. A scaffold-free cell aggregate as used herein is meant to distinguish the cell aggregate from engineered tissue constructs in which the cells are seeded or embedded into a continuous polymer matrix or scaffold, such as a hydrogel, that encompasses the cells. In contrast, the scaffold-free cell aggregate include discontinuous, dispersed insoluble native collagen fibers that can support adhesion and growth of cells, provide mechanical strength to the cell aggregate as well as cell aggregate size and/or thickness.

By self-assembled, it is meant that the cells can aggregate or assemble spontaneously or by themselves and without mechanical manipulation while in culture into cell aggregates having defined shapes, such as sheets. However, mechanical manipulation may be applied to facilitate the process. Such assembly can be caused by cell-cell interactions, interactions with the insoluble native collagen fibers, or formation of a self-secreted extracellular matrix that can bind to or permit the adhesion of cells in the aggregate.

The scaffold-free, high density cell aggregate can include a population of cells and a plurality of insoluble native collagen fibers that are dispersed with the cells within the cell aggregate.

The cells used to form the cell aggregate can be autologous, xenogeneic, allogeneic, and/or syngeneic. Where the cells are not autologous, it may be desirable to administer immunosuppressive agents in order to minimize immunorejection. The cells employed may be primary cells, expanded cells, or cell lines, and may be dividing or non-dividing cells. Cells may be expanded ex vivo prior to mixing with the insoluble native collagen fibers. For example, autologous cells can be expanded in this manner if a sufficient number of viable cells cannot be harvested from the host subject. Alternatively or additionally, the cells may be pieces of tissue, including tissue that has some internal structure. The cells may be primary tissue explants and preparations thereof, cell lines (including transformed cells), or host cells.

In some embodiments, the cell can be an undifferentiated or substantially differentiated progenitor cell. In other embodiments, the progenitor cell can be an adult stem cell or adult cancer cell. The stem cell, such as an adult stem cell or adult cancer cell, can be isolated from animal or human tissues. The cell used for the production of the cell sheet can be autologous or allogeneic. In the embodiments described herein, the stem cell can be isolated from, but not limited to, tendon/ligament tissue, bone morrow, adipose tissue or dental pulp. The cell aggregate can include at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% cells based on the total volume of the cell aggregate.

In various embodiments, the insoluble native collagen fibers provided in the cell aggregate can be crosslinked, either before or after being provided in the cell aggregate. In some embodiments, the use of crosslinked collagen fibers imparts improved characteristics such as: mechanical strength (for example, suturability, compression, tension) and biodurability (for example, resistant to enzymatic and hydrolytic degradation). By increasing the percentage of cross-links, for example, the degradation rate of the insoluble native collagen fibers can be decreased. Additionally, the compressive stiffness of the insoluble native collagen fibers can be increased by increasing the percentage of cross-links.

Crosslinking may be accomplished using several different crosslinking agents, or techniques (for example, thermal dehydration, EDC, aldehydes (e.g., formaldehyde, gluteraldehyde), natural crosslinking agents, such as genipin or proanthocyanidin, polyphenol, inorganic agents, such as iron, titanium, chromium, zirconium, aluminum ions and their complexes, and combinations thereof). Each type of crosslinking agent/technique or combinations thereof imparts diverse mechanical and biological properties on the material. These properties are created through the formation of unique chemical bonds that stabilize the insoluble native collagen fibers in the cell aggregate.

The insoluble native collagen fibers can also be modified to enhance cell function, such as differentiation, and/or enhance or accelerate tissue formation as promote cell adhesion. For example, the insoluble native collagen fibers can include at least one attachment molecule to facilitate attachment of at least one cell thereto. The attachment molecule can include a polypeptide or small molecule, for example, and may be chemically immobilized onto insoluble native collagen fibers to facilitate cell attachment. Examples of attachment molecules can include fibronectin or a portion thereof, or a portion thereof, polypeptides or proteins containing a peptide attachment sequence (e.g., arginine-glycine-aspartate sequence) (or other attachment sequence), enzymatically degradable peptide linkages, cell adhesion ligands, growth factors, degradable amino acid sequences, and/or protein-sequestering peptide sequences.

In some embodiments, the cell aggregate can include at least one, two, three, or more bioactive agent(s) that is capable of modulating a function and/or characteristic of a cell. For example, the bioactive agent may be capable of modulating a function and/or characteristic of a cell that is dispersed with the insoluble native collagen fibers, nanoparticles and/or microparticles. Alternatively or additionally, the bioactive agent may be capable of modulating a function and/or characteristic of an endogenous cell surrounding a tissue construct formed of the cell aggregate implanted in a tissue defect.

In some embodiments, the at least one bioactive agent can include polynucleotides and/or polypeptides encoding or comprising, for example, transcription factors, differentiation factors, growth factors, and combinations thereof. The at least one bioactive agent can also include any agent capable of promoting tissue formation (e.g., bone and/or cartilage), destruction, and/or targeting a specific disease state (e.g., cancer). Examples of bioactive agents include chemotactic agents, various proteins (e.g., short term peptides, bone morphogenic proteins, collagen, glycoproteins, and lipoprotein), cell attachment mediators, biologically active ligands, integrin binding sequence, various growth and/or differentiation agents and fragments thereof (e.g., EGF), HGF, VEGF, fibroblast growth factors (e.g., bFGF), PDGF, insulin-like growth factor (e.g., IGF-I, IGF-II) and transforming growth factors (e.g., TGF-β I-III), parathyroid hormone, parathyroid hormone related peptide, bone morphogenic proteins (e.g., BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-13, BMP-14), sonic hedgehog, growth differentiation factors (e.g., GDF5, GDF6, GDF8), recombinant human growth factors (e.g., MP-52 and the MP-52 variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-1, CDMP-2, CDMP-3), small molecules that affect the upregulation of specific growth factors, tenascin-C, hyaluronic acid, chondroitin sulfate, fibronectin, decorin, thromboelastin, thrombin-derived peptides, heparin-binding domains, heparin, heparin sulfate, polynucleotides, DNA fragments, DNA plasmids, MMPs, TIMPs, interfering RNA molecules, such as siRNAs, miRNAs, DNA encoding for an shRNA of interest, oligonucleotides, proteoglycans, glycoproteins, and glycosaminoglycans.

It will be appreciated at least one bioactive agent can be incorporated on or within insoluble native collagen fibers. The insoluble native collagen fibers can differentially or controllably release the at least one bioactive agent or be taken up (e.g., via endocytosis) by at least one cell to modulate the function and/or characteristic of the cell. The at least one bioactive agent may be at least partially coated on the surface of the at least one insoluble native collagen fiber. Alternatively, the at least one bioactive agent may be dispersed, incorporated, and/or impregnated within the insoluble native collagen fibers. For example, a bioactive agent comprising a DNA plasmid (e.g., a plasmid encoding BMP-2) can be coated onto the surface of insoluble native collagen fibers. It will be appreciated that one or more of the same or different bioactive agents can be incorporated on or within the insoluble native collagen fibers.

In some embodiments, a bioactive agent can comprise an interfering RNA or miRNA molecule incorporated on or within insoluble native collagen fibers or dispersed on or within the cell aggregate. The interfering RNA or miRNA molecule can include any RNA molecule that is capable of silencing an mRNA and thereby reducing or inhibiting expression of a polypeptide encoded by the target mRNA. Alternatively, the interfering RNA molecule can include a DNA molecule encoding for a shRNA of interest. For example, the interfering RNA molecule can comprise a short interfering RNA (siRNA) or microRNA molecule capable of silencing a target mRNA that encodes any one or combination of the polypeptides or proteins described above. The at least one microparticle can differentially or controllably release the at least one interfering RNA molecule or be taken up (e.g., via endocytosis) by at least one cell to modulate a function and/or characteristic of the cell.

The type, distribution, size, and/or crosslinking of the insoluble native collagen fibers can also be modified or configured to differentially, controllably, spatially, and/or temporally release at least one bioactive agent in the cell aggregate

In some embodiments, the cell aggregate can further include various nanoparticles and/or microparticles dispersed with the cells and insoluble native collagen fibers. Incorporation of the nanoparticles and/or microparticles in the cell aggregate can also improve the mechanical properties (e.g., compressive equilibrium modulus and tensile strength) of the cell aggregate and enable more uniform extracellular matrix deposition compared to cell aggregates without the nanoparticles and/or microparticles. The nanoparticles and/or microparticles that are dispersed in the cell aggregate can be formed from a biocompatible and biodegradable material that is capable of improving properties of the cell aggregate and which upon degradation is substantially non-toxic. The microparticles can have a diameter less than 1 mm and typically between about 1 nm and about 200 μm, e.g., about 20 μm to about 100 μm. The nanoparticles and/or microparticles can include nanospheres, nanocapsules, microspheres, and microcapsules, and may have an approximately spherical geometry and be of fairly uniform size.

The nanoparticles and/or microparticles can include nanospheres and/or microspheres that have a homogeneous composition as well as nanocapsules and/or microcapsules, which include a core composition (e.g., a bioactive agent) distinct from a surrounding shell. For the purposes of the present invention, for the purposes of the present invention, the terms “nanosphere,” “nanoparticle,” and “nanocapsule” may be used interchangeably, and the terms “microsphere,” “microparticle,” and “microcapsule” may be used interchangeably.

In some embodiments, the nanoparticles and/or microparticles can be formed from a biocompatible and biodegradable polymer. Examples of biocompatible, biodegradable polymers include natural polymers, such fibrin, gelatin, glycosaminoglycans (GAG), poly (hyaluronic acid), poly(sodium alginate), alginate, hyaluronan, and agarose. Other examples of biocompatible, biodegradable polymers are poly(lactide)s, poly(glycolide)s, poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s, polycaprolactone, polycarbonates, polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters, polyacetyls, polycyanoacrylates, polyetheresters, poly(dioxanone)s, poly(alkylene alkylate)s, copolymers of polyethylene glycol and poly(lactide)s or poly(lactide-co-glycolide)s, biodegradable polyurethanes, and blends and/or copolymers thereof.

Still other examples of materials that may be used to form nanoparticles and/or microparticles can include chitosan, poly(ethylene oxide), poly (lactic acid), poly(acrylic acid), poly(vinyl alcohol), poly(urethane), poly(N-isopropyl acrylamide), poly(vinyl pyrrolidone) (PVP), poly (methacrylic acid), poly(p-styrene carboxylic acid), poly(p-styrenesulfonic acid), poly(vinylsulfonicacid), poly(ethyleneimine), poly(vinylamine), poly(anhydride), poly(L-lysine), poly(L-glutamic acid), poly(gamma-glutamic acid), poly(carprolactone), polylactide, poly(ethylene), poly(propylene), poly(glycolide), poly(lactide-co-glycolide), poly(amide), poly(hydroxylacid), poly(sulfone), poly(amine), poly(saccharide), poly(HEMA), poly(anhydride), polyhydroxybutyrate (PHB), copolymers thereof, and blends thereof.

In some embodiments, the biocompatible and biodegradable polymer is a biodegradable hydrogel, such as gelatin. The biodegradable hydrogel can include a plurality of natural macromers that can be cross-linked using a cross-linking agent to provide a plurality of cross-links. Various sugar derivatives, such as glyoxal, D-ribose, or genipin can be used to cross-link the hydrogel. Other cross-linking agents, such as glutaraldehyde, can also be used. Concentrations of the crosslinking agent as well as time and temperature used for crosslinking can be varied to obtain the optimal results

The nanoparticles and/or microparticles can also be formed from inorganic materials, such as calcium phosphate materials including mineralite, carbonated nano-apatite, calcium phosphate based mineralite, tri-calcium phosphate, octa-calcium phosphate, calcium deficient apatite, amorphous calcium phosphate, hydroxyapatite, substitute apatite, carbonated apatite-like minerals, highly substituted carbonated apatites or a mixture thereof. Calcium phosphate nanoparticles and/or microparticles can have an average particle size of between about 1 nm and about 200 μm. It will be appreciated that smaller or larger calcium phosphate nanoparticles and/or microparticles may be used. The calcium phosphate nanoparticles and/or microparticles can have a generally spherical morphology and be of a substantially uniform size or, alternatively, may be irregular in morphology. Calcium phosphate nanoparticles and/or microparticles may be complexed with surface modifying agents to provide a threshold surface energy sufficient to bind material (e.g., bioactive agents) to the surface of the microparticle without denaturing the material. Non-limiting examples of surface modifying agents can include basic or modified sugars, such as cellobiose, carbohydrates, carbohydrate derivatives, macromolecules with carbohydrate-like components characterized by an abundance of —OH side groups and polyethylene glycol.

It will be appreciated at least one bioactive agent can also be incorporated on or within the nanoparticles and/or microparticles. The nanoparticles and/or microparticles similar to the collage fibers can differentially or controllably release the at least one bioactive agent or be taken up by at least one cell to modulate the function and/or characteristic of the cell. The at least one bioactive agent may be at least partially coated on the surface of the at least one of the nanoparticles and/or microparticles. Alternatively, the at least one bioactive agent may be dispersed, incorporated, and/or impregnated within the nanoparticles and/or microparticles.

In some embodiments, the cell aggregate can include a plurality of first nanoparticles and/or microparticles that can include or release one or more first bioactive agent(s) and a plurality of second nanoparticles and/or microparticles that can include or release one or more second bioactive agent(s). The one or more first bioactive agents and the one or more second bioactive agents may comprise the same or different agents. The one or more first bioactive agents and the one or more second bioactive agents can be differentially, sequentially, and/or controllably released from the first nanoparticles and/or microparticles and second nanoparticles and/or microparticles to modulate a different function and/or characteristic of a cell. It will be appreciated that the one or more first bioactive agents can have a release profile that is the same or different from the release profile of the one or more second bioactive agents from the first nanoparticles and/or microparticles and the second nanoparticles and/or microparticles. Additionally, it will be appreciated that the first nanoparticles and/or microparticles can degrade or diffuse before the degradation or diffusion of the second nanoparticles and/or microparticles or allow for an increased rate of release or diffusion of the one or more first bioactive agents compared to the release of the one or more second bioactive agents. The first and second nanoparticles and/or microparticles may be dispersed uniformly on or within the cell aggregate or, alternatively, dispersed such that different densities of the first nanoparticles and/or microparticles and second nanoparticles and/or microparticles are localized on or within different portions of the cell aggregate.

In some embodiments, the scaffold-free cell aggregate can be formed by combining the insoluble native collagen fibers with the cells and optionally nanoparticles and/or microparticles and then suspending the cells, insoluble native collagen fibers and optional nanoparticles and/or microparticles in a culture medium. The cell aggregate can also include a growth factor, such as TGFB1, that can be loaded in the collagen fibers and/or nanoparticles and/or microparticles and controllably released from the collagen fibers and/or nanoparticles and/or microparticles.

The cells can include any totipotent stem cell, pluripotent stem cell, or multipotent stem cell, immortalized cell, cancer cell, cancer stem cell, and/or differentiated cell. Progenitor cells can include autologous cells; however, it will be appreciated that xenogeneic, allogeneic, or syngeneic cells may also be used. The progenitor cells employed may be primary cells, expanded cells or cell lines, and may be dividing or non-dividing cells. The cells can be derived from any desired source. For example, the cells may be derived from primary tissue explants and preparations thereof, cell lines (including transformed cells) that have been passaged once (P1), twice (P2), or even more times, or host cells (e.g., human hosts). Any known method may be employed to harvest cells for use in the present invention. For example, mesenchymal stem cells, which can differentiate into a variety of mesenchymal or connective tissues (e.g., adipose tissue, osseous tissue, cartilaginous tissue, elastic tissue, and fibrous connective tissues), can be isolated according to the techniques disclosed in U.S. Pat. No. 5,486,359 to Caplan et al. and U.S. Pat. No. 5,226,914 to Caplan et al., the entireties of which are hereby incorporated by reference. In one example, the population of cells can comprise a population of human mesenchymal stem cells. In another example, the population of cells can comprise a population of human adipose derived stem cells.

The culture medium may include, for example, high-glucose DMEM supplemented with dexamethasone, ascorbate-2-phosphate, sodium pyruvate, and a premix of insulin, transferrin and selenium (ITS). By way of example, the medium may include high-glucose DMEM containing about 100 mM sodium pyruvate, about 80 μM ascorbate-2-phosphate, about 100 nM dexamethasone, and about 1% ITS. Additional medium components may include L-Glutamine, DMEM non-essential amino acid solution, and/or an antibiotic/antimycotic.

The insoluble native collagen fibers and optional nanoparticles and/or microparticles may be dispersed with cells in the suspension in a substantially uniform manner. The suspension can be provided in a culture vessel or chamber including but not limited to a tube, multiwall plate, transwell membrane, or bioreactor. The density at which the cells are seeded into the culture chamber can be, for example, about 1×10³ cells/mL to about 100×10⁶ cells/mL.

It will be appreciated that growth factors can be added to the medium to enhance or stimulate cell growth. Examples of growth factors include transforming growth factor-β (TGF-β) (e.g., TGF-β1 or TGF-β3), platelet-derived growth factor, insulin-like growth factor, acid fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor, hepatocytic growth factor, keratinocyte growth factor, and bone morphogenic protein. It will also be appreciated that other agents, such as cytokines, hormones (e.g., parathyroid hormone, parathyroid hormone-related protein, hydrocortisone, thyroxine, insulin), fatty acids (e.g., Omega-3 fatty acids such as α18:3 linolenate), and/or vitamins (e.g., vitamin D), may also be added or removed from the serum-free medium to promote cell growth.

The cells, insoluble native collagen fibers, and optional nanoparticles and/or microparticles can be cultured at a temperature and atmosphere effective to promote formation of cell aggregate. For example, the cells may be cultured at a temperature of about 37° C. in an atmosphere of about 5% carbon dioxide at an about 90% to about 95% humidity. The oxygen percentage can be varied from about 1% to about 21%. Typically, the cells can be cultured for about 1 to about 10 weeks.

As a result of culturing the cells with the insoluble native collagen fibers and optional nanoparticles and/or microparticles, a mechanically robust cell aggregate can be formed that can readily shaped, transferred, and/or manipulated to form the tissue construct. In some embodiments, the cell aggregate can comprise a single layer or sheet of tissue differentiated or substantially differentiated cells that are dispersed within an endogenously produced extracellular matrix with the completely or partially degraded collagen fibers and microspheres. The shape of the cell aggregate is not limited can vary depending on the culture vessel utilized and the culturing conditions.

The cell aggregate can be harvested from the by, for example, lifting the layer of cell aggregate out of the culture chamber. The cell aggregate so formed can be shaped, molded, or configured into a variety of tissue constructs that can be used in various biomedical applications, including tissue engineering, drug discovery applications, and regenerative medicine. In one example, a tissue construct comprising the cell aggregate can be used to promote tissue growth in a subject by administering the tissue construct to a target site. The target site can comprise a tissue defect (e.g., cartilage and/or bone defect) in which promotion of new tissue (e.g., cartilage and/or bone) is desired. The target site can also comprise a diseased location (e.g., tumor). Methods for identifying tissue defects and disease locations are known in the art and can include, for example, various imaging modalities, such as CT, MRI, and X-ray.

The tissue defect can include a defect caused by the destruction of tissue or bone or cartilage. For example, one type of cartilage defect can include a joint surface defect. Joint surface defects can be the result of a physical injury to one or more joints or, alternatively, a result of genetic or environmental factors. Most frequently, but not exclusively, such a defect will occur in the knee and will be caused by trauma, ligamentous instability, malalignment of the extremity, meniscectomy, failed aci or mosaicplasty procedures, primary osteochondritis dessecans, osteoarthritis (early osteoarthritis or unicompartimental osteochondral defects), or tissue removal (e.g., due to cancer). Examples of bone defects can include any structural and/or functional skeletal abnormalities. Non-limiting examples of bone defects can include those associated with vertebral body or disc injury/destruction, spinal fusion, injured meniscus, avascular necrosis, cranio-facial repair/reconstruction (including dental repair/reconstruction), osteoarthritis, osteosclerosis, osteoporosis, implant fixation, trauma, and other inheritable or acquired bone disorders and diseases.

Tissue defects can also include cartilage defects. Where a tissue defect comprises a cartilage defect, the cartilage defect may also be referred to as an osteochondral defect when there is damage to articular cartilage and underlying (subchondral) bone. Usually, osteochondral defects appear on specific weight-bearing spots at the ends of the thighbone, shinbone, and the back of the kneecap. Cartilage defects in the context of the present invention should also be understood to comprise those conditions where surgical repair of cartilage is required, such as cosmetic surgery (e.g., nose, ear). Thus, cartilage defects can occur anywhere in the body where cartilage formation is disrupted, where cartilage is damaged or non-existent due to a genetic defect, where cartilage is important for the structure or functioning of an organ (e.g., structures such as menisci, the ear, the nose, the larynx, the trachea, the bronchi, structures of the heart valves, part of the costae, synchondroses, enthuses, etc.), and/or where cartilage is removed due to cancer, for example.

After identifying a target site, such as a cranio-facial cartilage defect of the nose, the tissue construct can be administered to the target site by, for example, implantation into the tissue defect. Prior to implantation, the tissue construct comprising the cell aggregate can be formed into the shape of the tissue defect using tactile means. Alternatively, the tissue construct may be formed into a specific shape prior to implantation into the subject.

The tissue construct may be attached to the target site using, for example, adhesive materials, such as bioadhesives, sutures, staples, and/or a membrane covering. Examples of adhesive materials include calcium phosphate-based pastes (e.g., αBMM), fibrin-based glues, transglutaminase, and chemical cross-linking agents (e.g., 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride).

After injecting implanting the tissue construct into the subject, the cells of the tissue construct can express growth and/or differentiation factors, and/or promote progenitor cell expansion and differentiation. Additionally, the presence of the tissue construct in the tissue defect may promote migration of endogenous cells surrounding the tissue defect into the tissue construct. Moreover, the tissue construct can secrete factors that affect surrounding cells in a trophic manner. This can act to improve healing/regeneration, immunomodulate or treat disease states.

In some embodiments, a heterogenous cell aggregate or tissue construct can be formed that includes defined regions or portion (e.g., layers) of differing or similar cell aggregate materials. The differing regions or portions of the heterogenous cell aggregate or tissue construct can be provided or formed with or without insoluble native collagen fibers a and/or nanoparticles and/or microparticles and can have similar or different properties to vary the properties of the tissue construct for particular tissue engineering applications.

In some embodiments, a heterogenous cell aggregate can be formed by seeding a first mixture of insoluble native collagen fibers, cells, and optional nanoparticles and/or microparticles in a culture chamber and then seeding at least a second mixture of cells and insoluble native collagen fibers, cells, and optional nanoparticles and/or microparticles over, around, within, and/or along select portions of the seeded first mixture of insoluble native collagen fibers, cells, and optional nanoparticles and/or microparticles. The first mixture of insoluble native collagen fibers, cells, and optional nanoparticles and/or microparticles can include the same or different type, concentration, amount, and/or distribution, of insoluble native collagen fibers, cells, and optional nanoparticles and/or microparticles and/or potentially bioactive agents as the second mixture of insoluble native collagen fibers, cells, and optional nanoparticles and/or microparticles to vary the compositions and properties of the tissue construct for particular tissue engineering applications. It will be appreciated that the first mixture and/or second mixture may be free of cells. For example, a heterogenous cell aggregate can be formed by seeding a first mixture of insoluble native collagen fibers, cells, and optional nanoparticles and/or microparticles in a culture chamber and then seeding a mixture of cells that is free of insoluble native collagen fibers, and nanoparticles and/or microparticles over, around, within, and/or along select portions of the seeded first mixture of nanoparticles and/or microparticles and cells.

In other embodiments, a heterogenous cell aggregate or tissue construct that includes defined regions or portion (e.g., layers) of differing materials can be formed by layering portions or sheets of cell aggregates described herein can layered to from a heterogenous or multilayer tissue construct. For example, a first portion of a sheet of cell aggregate may be folded over onto a second portion of the sheet. An optional load, such as a compressive, load can then be applied to the folded construct for an amount of time (e.g., about 1 week) effective to promote integration of the layers.

The multilayer cartilage tissue construct so formed may be removed from the culture vessel and applied to an articular surface. Additionally, it should be appreciated that the thickness of the multilayer cartilage tissue construct may be adjusted as needed by adding or removing layers. For example, a plurality of layers may be sandwiched, adhered, or mechanically manipulated by, for example, compression, tension, hydrostatic loading, or shear loading, and formed into a multilayer cartilage tissue construct having a thickness of about 1 mm to about 4 mm or greater.

In another method, at least two tissue constructs can be formed from similar and/or substantially different cell aggregates may be layered on one another and then allowed to integrate or adhere and/or mechanically manipulated, by, for example, compression, tension, hydrostatic loading, or shear loading. In still other embodiments, the cell aggregates described herein can be combined with or adhered to other tissue constructs to form a heterogenous tissue construct.

In other embodiments, the DNA or cells in the cell aggregate can be removed or lysed to provide an acellular tissue construct that includes the extracellular matrix so formed and, potentially, the partially or completely degraded insoluble native collagen fibers. Removal may be achieved by, for example, detergent treatment, (e.g., SDS treatment) treatment with DNase and RNase, and/or freeze/thaw cycles. The acellular tissue construct can then be used alone for tissue engineering application or in combination with other cell types or growth factors for the promotion of tissue repair. The acellular tissue construct can be used as an acellular biomaterial for tissue engineering application similar to the above after decellularization. When used alone, the acellular tissue can be used to prevent or repair tissue defects, enhance host cell attachment, infiltration, differentiation, extension, and proliferation. The acellular tissue construct as a decellularized product can be used together with other known bioactive agents and cell types for the promotion of tissue repair.

The following examples are for the purpose of illustration only and are not intended to limit the scope of the claims, which are appended hereto.

Example

In nature, cells reside in ECMs that are highly ordered and hierarchical. Such structure regulates cell morphology, signaling, gene expression and apoptosis. As one of the most abundant ECMs, collagen supports a variety of cell types in most of human organs and tissues. Collagen molecules assemble into fibrils with the diameter of hundred nanometers. These fibrils bundled to fibers with the diameter of about 5-20 μm. Collagen fibers but not fibrils, provide sufficient mechanical, chemical and biological support to cells. Cells reside, proliferate, differentiate and migrate within the gaps of fibers. Unfortunately, few scaffolds have been developed to imitate the natural collagen fiber for 3D cell growth and tissue engineering. Re-assembly of collagen molecules in vitro often results in fibrils but not fibers, which is usually known as collagen gel. Although such matrix has been widely used and have proven to support cell growth, due to the fine size of the fibrils, the direct contact of cells is inhibited as cells are surrounded by those tiny fibrils. Reducing the fibril density increase the pore size for cell-cell interaction but at the cost of declined mechanical strength of the matrix.

Since collagen fibers are one of the major natural ECM to support cell growth, we hypothesize that directly isolating these collagen fibers from tissues potentially provides a microenvironment for resided cells mimicking the natural conditions. In this example, we enzymatically dissociated collagen rich tissue scaffold to independent collagen fibers. Obtained collagen fibers are flexible, allowing a variety types of cells to proliferate and migrate. Cell-cell interactions are also maintained, evident by vasculogenesis of endothelial cells almost completely relying on the interactions with their adjacent MSCs.

At the first step, decellularized, purified and ground tissue powder, such as skin tissue, was chosen to extract the dispersible collagen fibers. The skin powders were soaked into 1 M sulfuric acid containing 1 M NaCl for overnight to loosen the fibrous structure and remove nucleic acids. After neutralization, the slurry was dissociated with pepsin under 4° C. However, instead of fully degrading the tissue scaffold to soluble collagen molecules, the dissociation was controlled to a degree that massive fiber bundles were untied but few were degraded to collagen fibrils or free molecules. Sequentially, collagen fibers were purified by gradual centrifugations to remove bulky materials, fibrils and dissolved collagen molecules. Obtained collagen fibers feature about 5-20 μm in diameter and hundreds microns in length. The fibers contain mostly collagen type I, and a little collagen type III, according to proteomic analysis. The fibers are highly flexible and can be easily reshaped in molds in the presence of external force. For example, the fibers form a soft but robust sheet in cell culture plates after simple centrifugation.

To verify the biocompatibility, two cell lines, including NIH/3T3 and HeLa cells, representing mouse and human sources respectively, were mixed individually with collagen fibers and cultured in 24 well plate as a sheet with the thickness of about 200 μm after centrifugation. As shown in FIG. 2, cells successfully resided in collagen fiber platform after 1 day. DNA quantification indicated that cells were able to proliferate in the collagen fiber platform for at least 7 days. Similarly, human mesenchymal stem cells (hMSCs) obtained from bone marrow of healthy donor showed similar viability in the collagen fiber platform (FIG. 2E). LSCM results revealed that hMSCs proliferated in the platform during one-week culture. Since collagen fiber platform is composed of entangled fibers that is similar to randomly stacked ropes, there are numerous spaces and gaps within the platform for cell growth and migration.

Due to its unique porous and cytocompatible properties, it is expected that this platform supports multiple type cell interactions for more complex tissue building. hMSCs are important pericytes and they help endothelial cells survive and form vasculatures through paracrine pathway. Therefore, we co-cultured hMSCs and HUVEC in the collagen fiber platform for the observation of vasculogenesis. To maximize the influence of hMSC-HUVEC interaction, the cells were cultured in DMEM supplied with 10% FBS and 10 ng/mL FGF-2 that is insufficient for HUVEC alone to survive. However, when cultured together in the collagen fiber platform, HUVEC survived and started to form vasculature within 3 days. After 7 days, the vasculature greatly intensified. To verify the necessity of cell-cell contact, we placed hMSCs on the top layer of transwell plate and the HUVECs on the bottom layer. After 3 days, no HUVEC survived. This result proved that direct contact with hMSCs is vital for the survive and vasculature formation of HUVECs. Next, we converted the culture conditions to traditional 3D platforms, including 10% GelMA and collagen gels. GelMA is derived from gelatin, a denatured form of collagen. After UV crosslinking, GelMA form robust hydrogels that is biocompatible to a variety of cells. However, GelMA confines cells from migration with its steady structure but is permeable for nutrient and protein signals due to its porous framework. After 3 or 7-day culture, no vasculature was formed in GelMA. In contrast, few HUVECs survived in the gel although hMSCs were viable. Similarly, vasculature was not formed in traditional collagen gel up to 7 days. These results demonstrated that strong MSC-HUVEC interaction must take place for HUVEC growing and vascularization. Compared with 2D culture, vasculature of the endothelial cells was not efficiently formed in traditional scaffold based 3D culture methods, with co-culturing hMSCs and HUVEC. However, culturing both cell types in collagen fiber platform, vasculature started to form within 3 days. Taken together, collagen fiber platform enhances cell-cell direct interactions due to the facile migration ability of cells in this platform.

We then tested the functions of precrosslinked collagen fibers. To perform the crosslinking, collagen fibers were treated with certain amount of crosslinking reagent, such as 0.05%-0.2% glutaraldehyde in PBS for 10 min, and neutralized by glycine or serum proteins for another 10 min. After thorough washing, shear stress of collagen fibers was characterized using rheometer. As shown in FIG. 4A, with the increase of crosslinking percentage, the shear modulous of collagen fiber increases. After incubation in 37° C. for overnight, the shear modulus of both uncrosslinked and crosslinked collagen fibers significantly increased. This result suggested that the mechanical properties of collagen fiber are tunable through chemical modification. After mixing with hMSCs and endothelial cells for coculture described as above, the vasculogenesis of endothelial cells was evaluated (FIG. 4B, C). Apparently, with the increase of the crosslinking, the vasculogenesis of endothelial cells declines. This result demonstrated that with tailored chemical and mechanical properties of collagen fibers, behavior of lodged cells can be regulated.

From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims. All references, publications, and patents cited in the present application are herein incorporated by reference in their entirety. 

Having described the invention, we claim:
 1. A method of forming an insoluble native collagen fiber additive; the method comprising; soaking decellularized tissue powder, which includes native colleen fiber, in acidic or basic, salt solution for an amount of time effective to loosen the fibrous structure and remove nucleic acids and forming a slurry that includes insoluble collagen fibers; enzymatically dissociating the insoluble collagen fibers in the slurry to a degree that bundle of insoluble collagen fibers are loosened and a native architecture of the insoluble collagen fibers is maintained with minimal generation of collagen fibril, isolating insoluble native collagen fibers from the dissociated slurry.
 2. The method of claim 1, wherein the insoluble collagen fibers are enzymatically dissociated with pepsin.
 3. The method of claim 1, wherein the acidic, salt solution includes 1 M sulfuric acid and 1 M NaCl and the basic salt solution includes 1 M NaOH or Ca(OH)₂ and 1 M NaCl.
 4. The method of claim 1, wherein the insoluble native collagen fibers are isolated by separating the insoluble native collagen fibers from dispersed collagen fibrils, soluble collagen, and non-collagen material in the dissociated slurry.
 5. The method of claim 1, wherein the insoluble native collagen fibers have an average diameter of about 1 μm to about 200 μm and average length of about 100 μm to about 3 mm.
 6. The method of claim 1, wherein the decellularized tissue powder comprises decellularized ground skin, fascia, intestine, tendon, bladder, trachea, heart, lung, liver, kidney, spleen and/or pancreas powder.
 7. Insoluble native collagen fibers formed by the method of claim
 1. 8. The insoluble native collagen fibers of claim 7, being substantially free of unbound soluble collagen molecules or unbound collagen fibrils.
 9. A cell aggregate comprising: a plurality of dispersed cells and a plurality of dispersed insoluble native collagen fibers, the collagen fibers supporting adhesion and/or growth of cells in the cell aggregate, the insoluble native collagen fibers having an average diameter of about 1 μm to about 200 μm and average length of about 100 μm to about 3 mm.
 10. The cell aggregate of claim 9, be scaffold-free.
 11. The cell aggregate of claim 9, being self-assembled.
 12. The cell aggregate of claim 9, the insoluble native collagen fibers comprising collagen type I, collagen type II, collagen type III, collagen type IV or combinations thereof.
 13. The cell aggregate of claim 9, the insoluble native collagen fibers being crosslinked.
 14. The cell aggregate of claim 9, further comprising at least one bioactive agent.
 15. The cell aggregate of claim 14, the at least bioactive agent being incorporated in or physically associated with the insoluble native collagen fibers
 16. The cell aggregate of claim 9, further comprising a plurality of nanoparticles and/or microparticles, the bioactive agent being incorporated in or physically associated with the nanoparticles and/or microparticles and released with a defined or controlled release profile from the nanoparticles and/or microparticles.
 17. The cell aggregate of claim 9, the cell aggregate comprising undifferentiated and/or substantially differentiated cells.
 18. The cell aggregate of claim 11, the cells being at least about 10%, by volume of the cell aggregate based on the total volume of the cell aggregate.
 19. The cell aggregate of claim 18, the cells comprising immortalized cells, cancer cells, cancer stem cells, progenitor cells, or stem cells.
 20. The cell aggregate of claim 11, including defined regions or portions having differing types or concentrations of cells and/or insoluble native collagen fibers. 